Botulinum toxin, the most poisonous substance known, is one of the top six 'Category A' biological agents. This agent is extremely potent, easy to produce and transport, and has high potential for morbidity and mortality, making it a major threat as a bio-weapon. Currently, there are no readily effective treatment measures for botulism, and death usually results from respiratory muscle paralysis. The diverse health consequences of botulinum toxin combined with the increased threat of bioterrorism underscore the critical need for exploratory research to identify molecular targets that may serve as potential sites for inhibition of toxin action at the clinically relevant target site, the mammalian neuromuscular junction (NMJ). The objective of this exploratory R21 application is to define the membrane target proteins that potentially mediate the selective uptake of botulinum toxin serotypes A and B by nerve terminals of the mammalian NMJ. To achieve this objective co-immunoprecipitation methodology will be combined with proteomic technology to identify proteins, which bind selectively to the toxins. A two-pronged approach will be followed that explores toxin-binding proteins in two different but complementary target tissue preparations, the mouse diaphragm and mouse spinal cord cells. For each target system, two separate sample preparations will be probed. These include 1) a synaptic protein-enriched crude membrane fraction developed in the PI's laboratory and 2) an early endosome-enriched fraction. Once potential membrane receptors for botulinum toxin serotypes A and B are characterized, it will be possible to develop and test toxin antagonists in future studies. If successful, similar protocols could then be applied to the identification of the membrane receptors for the remaining toxin serotypes. Collectively, the results of these studies will significantly impact clinical medicine by defining cellular targets at the NMJ that may serve as templates for the development of effective pharmacologic countermeasures to botulinum toxin exposure and safer toxin-like therapeutic agents.